Abstract Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), and was responsible for 10.4 million new infections and 1.8 million deaths in 2015. Current treatment regimens for TB are long (6 months or more), costly ($10,000-100,000), and have significant side effects. These facts reduce patient compliance, which leads to increased incidence of antibiotic resistance, spread of the disease, and severity of illness or death. This lengthy treatment is required because of the ability of Mtb to transition to a persistent state that is tolerant of antibiotics. Transition to persistence is proposed to occur through a complex metabolic remodeling in response to adverse conditions. The molecular mechanisms governing this transition are poorly understood, but conservation of redox homeostasis and oxidative phosphorylation via the electron transport chain (ETC) appears essential. While recent efforts in TB drug discovery and development have heavily focused on targeting the ETC, growing evidence indicates that plasticity in terminal oxidation is mitigating the efficacy of new inhibitors. Mtb expresses two terminal oxidases: (1) the primary cytochrome bc1:aa3 complex; and (2) the alternative cytochrome bd oxidase (Cyt-bd). This apparent functional redundancy appears to play an important role in respiratory chain flexibility, and Cyt-bd has been shown to protect the cell from two ETC inhibitors, BDQ and Q203. The alternative oxidase has also been implicated in protection from hypoxia, reductive stress and oxidative stress. However, the exact function of Cyt-bd remains unknown, and the lack of a model for its role in the ETC limits development of new therapies. We propose to systematically investigate the role of Cyt-bd in the adaptation of Mtb to adverse conditions and validate it as a novel drug target for combination therapy to enhance the bactericidal activity of current chemotherapeutic regimens. Our specific aims are: 1) to validate the biochemical roles of Cytochrome bd Oxidase. We will perform inverted membrane vesicle assays to determine its biochemical activity. 2) To define the functional role of Cyt-bd during adaptation to hypoxia, reductive stress, and oxidative stress. Whole cells will be challenged with each adverse condition, the response to which will be characterized by measuring redox pair ratios relevant to oxidative phosphorylation. 3) To demonstrate that inhibition of Cyt-bd sensitizes M. tuberculosis to existing chemotherapeutics. Cyt-bd will be tested as a drug target by determining existing chemotherapeutics whose bactericidal activity is enhanced by Cyt-bd inactivation. A mouse model of infection will be utilized to determine the in vivo potential of combination therapies that would target cytochrome bd oxidase. With the mentorship of Drs. Michael Berney and William R. Jacobs, I will be able to accomplish the goals for the proposed research to characterize, understand and validate the alternative terminal oxidase as a drug target to enhance the efficacy of current chemotherapy regimens. I will acquire new skills in biochemistry, membrane physiology, microbiology, and animal models. Furthermore, I will expand my training in communication skills, grant writing, manuscript writing, and mentoring to further enhance my success as a physician-scientist.